Embryonic Lizard Staining (Part 3)

Finally, after letting the skeletal prep de-stain in 2% KOH for 2 days, I was ready to take some real pics. The tissue cleared up a bit, allowing me to see the bones and cartilage better. 

(For reference on how to do skeletal preps with Alcian Blue and Alizarin Red, check out my previous blog posts Mouse Skeletal Prep, Lizard Embryo Pt I, Lizard Embryo Pt II.) Blue stains for cartilage and Red stains for bone. For reference on this green anole lizard embryo see Advanced Lizard Embryo and Hand Osteology.

Here is the stained skeletal prep of a green anole lizard embryo (Anolis carolinensis). 

Anole embryo skeletal prep, Alcian Blue and Alizarin Red staining solution (left lateral view)

Here is a view of the shoulder, arm and hand of the anole embryo. 

Skeleton of a green anole lizard embryo (left lateral view)

 Next is picture from below the jaw of the anole.

Green anole lizard embryo jaw and chest (ventral view)

You can make out the bottom of the skull bones, looking upward through the jaw from below.

Anole lizard jaw and skull bones (inferior view)

Developing sternum and clavicles of an anole lizard embryo (ventral view)

Here is what I think are the sterno-clavicular pre-cursors. The sternum is a bone at the midline of the chest. The clavicles run perpendicular to the A-P axis and articulate with the sternal bones. In reptiles. the sternum is more shield shaped, whereas in mammals the sternum is more elongated. The literature indicates that reptilien sternums are comprised mainly of cartilage. In the picture above and below, it appears that the sternum contains some calcium, indicative of bone. 

Anole lizard embryo sternal development (ventral view)

While the literature on embryonic anatomy is scarce, I did find an account of sternum/manubrium development in human embryonic studies. According to the paper, interclavicular mesenchyme condenses as "sternal bands" integrate into the mesenchyme. Neural crest cells, lateral plate mesoderm and somites contribute to the formation. Parts of the clavicle undergo endochondrial ossification (Rodríguez-Vázquez et al., 2012).

Next is a lateral view of the shoulder and pectoral girdle. Interestingly, there are "interclavicles" that span the neck line. The scapula extends backward towards the spine, but it is a very faint blue, which you can barely make out in the picture below.
 

Anole lizard embryo thoracic and shoulder girdle (right lateral view)

I used an agarose gel to position the anole lizard embryo on it's back.

Anolis carolinensis embryo skeletal prep (frontal view)

Here is a nice shot of the hands of the lizard embyro (Anolis carolinensis).

Anolis carolinensis embryo hands

Here is a close-up of the fingers. The metacarpals are the most proximal (closest) to the wrist's base. Stemming out from there is the next 3 set of bones: the proximal phalanges, the intermediate phalanges and the distal phalanges (fingertips).

Green anole lizard hand bones: metacarpals are stained red, indicating bone formation

Below is the elbow (humerus above and the radius and ulna below). Note that there is not bone at the elbow joint, as it is faintly dyed blue for cartilage.

Forearm of an anole lizard embryo: the humerus, radius and ulna (left lateral view)

Next, I tried to capture a lateral view of the vertebrae, though I think I reached the resolution limit of my microscope camera. 

Thoracic vertebrae and ribs of a lizard embryo (left lateral view)

Vertebrae of an Anolis carolinensis lizard embryo (left lateral view)

Below, you can see there is still a gap between the 2 lateral sides of each vertebrae. These are called dorsal fissures and they will close, as well as develop a neural spine. 
 

Vertebral column of an anolis lizard embryo (dorsal view)

Developing vertebrae of an anolis lizard embryo (dorsal view)

Dorsal fissures still adorn the vertebrae during this phase of development (in the pre-hatchling anole). 

Dorsal fissures of developing vertebrae in an anolis embryo (dorsal view)

The lizard embryo's vertebrae have yet to close. 

Dorsal fissure of a vertebra in the green anole lizard embryo (dorsal view)

Developing vertebrae and hemal arches of an anole lizard embryo

The vertebrae of the distal tail have odd processes protruding outwards. 

Caudal vertebrae of an anolis lizard embryo

Caudal vertebrae of an anolis lizard embryo

 Here are the vertebrae near the hip joint.

Lumbar and caudal vertebrae of an anolis lizard embryo (dorsal view)

Here is the femur (thigh) bone in focus, looking down from above.

Left femur of an anole lizard embryo (dorsal view)

Here are the bones (metatarsals) of the feet.

Metatarsals of an anole embryo (superior view)

 Here is one toe, with the nail cartilage in blue. 

Distal phalanges (toe) of an anole lizard embryo

Finally, a shot of the head which looks to me like anencephaly, or as if part of the brain and skull are missing. While the bones of the skull are very different and elongated in the anole lizard, there should be some bones present here, but are not. 

Rostral vertebral column of a green anole lizard embryo (left lateral)

Below is comparison of the lizard vertebrae and the mouse vertebrae. You can see that in the lizard Anolis carolinensis, the vertebrae have yet to close on the dorsal side, as indicated by fissures at each vertebrae. In the mouse, these fissures have closed and there is an extension of cartilage across the dorsal side.  While these two embryos are at different stages, respectively, there are major differences in vertebral anatomy. In the future, I should try to obtain equivalent stage embryos in lizard and mouse.

Upon finishing the chick, mouse and lizard skeletal preps, I will be conducting more comparisons of embryonic anatomy. 



References
Rodríguez-Vázquez JF, Verdugo-López S, Garrido JM, Murakami G, Kim JH. (2012). Morphogenesis of the manubrium of sternum in human embryos: a new concept. Anat Rec (Hoboken). 2013 Feb;296(2):279-89. doi: 10.1002/ar.22623. Epub 2012 Nov 19.


All embryos were treated ethically according to IUCAC protocol.
 

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